Changes in mammalian copper homeostasis during microbial infection.

Animals carefully control homeostasis of Cu, a metal that is both potentially toxic and an essential nutrient. During infection, various shifts in Cu homeostasis can ensue. In mice infected with Candida albicans, serum Cu progressively rises and at late stages of infection, liver Cu rises, while kidney Cu declines. The basis for these changes in Cu homeostasis was poorly understood. We report here that the progressive rise in serum Cu is attributable to liver production of the multicopper oxidase ceruloplasmin (Cp). Through studies using Cp-/- mice, we find this elevated Cp helps recover serum Fe levels at late stages of infection, consistent with a role for Cp in loading transferrin with Fe. Cp also accounts for the elevation in liver Cu seen during infection, but not for the fluctuations in kidney Cu. The Cu exporting ATPase ATP7B is one candidate for kidney Cu control, but we find no change in the pattern of kidney Cu loss during infection of Atp7b-/- mice, implying alternative mechanisms. To test whether fungal infiltration of kidney tissue was required for kidney Cu loss, we explored other paradigms of infection. Infection with the intravascular malaria parasite Plasmodium berghei caused a rise in serum Cu and decrease in kidney Cu similar to that seen with C. albicans. Thus, dynamics in kidney Cu homeostasis appear to be a common feature among vastly different infection paradigms. The implications for such Cu homeostasis control in immunity are discussed.

P2X7 receptor antagonism prevents IL-1ß release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy.

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1ß and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1ß, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1ß release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1ß release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.

Validation and Comparison of the Therapeutic Efficacy of Boron Neutron Capture Therapy Mediated By Boron-Rich Liposomes in Multiple Murine Tumor Models.

Boron neutron capture therapy (BNCT) was performed at the University of Missouri Research Reactor in mice bearing CT26 colon carcinoma flank tumors and the results were compared with previously performed studies with mice bearing EMT6 breast cancer flank tumors. Mice were implanted with CT26 tumors subcutaneously in the caudal flank and were given two separate tail vein injections of unilamellar liposomes composed of cholesterol, 1,2-distearoyl-sn-glycer-3-phosphocholine, and K[nido-7-CH3(CH2)15-7,8-C2B9H11] in the lipid bilayer and encapsulated Na3[1-(2`-B10H9)-2-NH3B10H8] within the liposomal core. Mice were irradiated 30 hours after the second injection in a thermal neutron beam for various lengths of time. The tumor size was monitored daily for 72 days. Despite relatively lower tumor boron concentrations, as compared to EMT6 tumors, a 45 minute neutron irradiation BNCT resulted in complete resolution of the tumors in 50% of treated mice, 50% of which never recurred. Median time to tumor volume tripling was 38 days in BNCT treated mice, 17 days in neutron-irradiated mice given no boron compounds, and 4 days in untreated controls. Tumor response in mice with CT26 colon carcinoma was markedly more pronounced than in previous reports of mice with EMT6 tumors, a difference which increased with dose. The slope of the dose response curve of CT26 colon carcinoma tumors is 1.05 times tumor growth delay per Gy compared to 0.09 times tumor growth delay per Gy for EMT6 tumors, indicating that inherent radiosensitivity of tumors plays a role in boron neutron capture therapy and should be considered in the development of clinical applications of BNCT in animals and man.

A Trimodal Closomer Drug-Delivery System Tailored with Tracing and Targeting Capabilities.

The construction and application of a unique monodisperse closomer drug-delivery system (CDDS) integrating three different functionalities onto an icosahedral closo-dodecaborane [B12 ](2-) scaffold is described. Eleven B-OH vertices of [closo-B12 (OH)12 ](2-) were used to attach eleven copies of the anticancer drug chlorambucil and the targeting vector glucosamine through a bifurcating lysine linker. The remaining twelfth vertex was used to attach a fluorescent imaging probe. The presence of multiple glucosamine units offered a monodisperse and highly water-soluble CDDS with a high payload of therapeutic cargo. This array enhanced the penetration of the drug into cancer cells by exploiting the overexpression of GLUT-1 receptors present on cancer cells. About 15-fold enhancement in cytotoxicity was observed for CDDS-1 against Jurkat cells, compared to CDDS-2, which lacks the GLUT-1 targeting glucosamine. A cytotoxicity comparison of CDDS-1 against colorectal RKO cells and its GLUT-1 knock-out version confirmed that GLUT-1 mediates endocytosis. Using fluorescent markers both CDDS-1 and -2 were traced to the mitochondria, a novel target for alkylating agents.

Synthesis and in vitro assessment of a bifunctional closomer probe for fluorine ((19)F) magnetic resonance and optical bimodal cellular imaging.

The design, synthesis and in vitro assessment of a bifunctional imaging probe for dual fluorine ((19)F) magnetic resonance spectroscopy ((19)F-MRS) and fluorescence detection is reported. Eleven copies of 3,5-bis(trifluoromethyl)phenyl and a single copy of a sulforhodamine-B were covalently attached to a closo-B12(2-)-core via suitable linkers. The (19)F-MRS and fluorescence imaging shows that, this novel bimodal imaging probe was readily taken up by the cells in vitro after co-incubation.

Synthesis of [closo-B12(OH)11NH3]-: a new heterobifunctional dodecaborane scaffold for drug delivery applications.

Effective utilization of [closo-B12H12](2-) derivatives in targeted drug delivery applications depends upon an efficient strategy to differentiate at least one of the 12 vertices on the B12(2-) core. Precursor molecules must also be able to withstand the initial harsh hydrogen peroxide treatment necessary for hydroxylation of the B-H vertices. We report here a method for preparation of the ammonio derivative [closo-B12(OH)11NH3](-) and also demonstrate its utility in construction of a targeted drug delivery scaffold. Treatment of the precursor [closo-B12H11NH3](-) with hydrogen peroxide gives the corresponding nitro derivative [closo-B12(OH)11NO2](2-) in good yield. The nitro group is easily reduced with hydrogen over a Raney nickel catalyst to produce [closo-B12(OH)11NH3](-). The 11 hydroxyl groups can then be readily converted to carbonates or carbamates. As a proof-of-principle of its utility as a drug delivery system, we used the resulting vertex-differentiated ammonio derivative to construct a platinated pro-drug possessing 11 copies of a carboplatin analogue conjugated to the B12(2-) core via carbamate linkage and a fluorescein molecule attached at the remaining vertex by an amide linkage. In vitro cytotoxicity assays demonstrated that activity of an untagged analog was similar to carboplatin against platinum-sensitive A459 cells and higher than carboplatin against platinum-resistant SK-OV-3 cells. Further fluorescence microscopy revealed that the fluorescein-tagged pro-drug localizes to the nuclei of A459 cells.

Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.

Pathologic angiogenesis mediated by abnormally polarized macrophages plays a central role in common age-associated diseases such as atherosclerosis, cancer, and macular degeneration. Here we demonstrate that abnormal polarization in older macrophages is caused by programmatic changes that lead to reduced expression of ATP binding cassette transporter ABCA1. Downregulation of ABCA1 by microRNA-33 impairs the ability of macrophages to effectively efflux intracellular cholesterol, which in turn leads to higher levels of free cholesterol within senescent macrophages. Elevated intracellular lipid polarizes older macrophages to an abnormal, alternatively activated phenotype that promotes pathologic vascular proliferation. Mice deficient for Abca1, but not Abcg1, demonstrate an accelerated aging phenotype, whereas restoration of cholesterol efflux using LXR agonists or miR-33 inhibitors reverses it. Monocytes from older humans with age-related macular degeneration showed similar changes. These findings provide an avenue for therapeutic modulation of macrophage function in common age-related diseases.

Boron neutron capture therapy demonstrated in mice bearing EMT6 tumors following selective delivery of boron by rationally designed liposomes.

The application of boron neutron capture therapy (BNCT) following liposomal delivery of a (10)B-enriched polyhedral borane and a carborane against mouse mammary adenocarcinoma solid tumors was investigated. Unilamellar liposomes with a mean diameter of 134 nm or less, composed of an equimolar mixture of cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine and incorporating Na3[1-(2'-B10H9)-2-NH3B10H8] in the aqueous interior and K[nido-7-CH3(CH2)15-7,8-C2B9H11] in the bilayer, were injected into the tail veins of female BALB/c mice bearing right flank EMT6 tumors. Biodistribution studies indicated that two identical injections given 24 h apart resulted in tumor boron levels exceeding 67 µg/g tumor at 54 h--with tumor/blood boron ratios being greatest at 96 h (5.68:1; 43 µg boron/g tumor)--following the initial injection. For BNCT experiments, tumor-bearing mice were irradiated 54 h after the initial injection for 30 min with thermal neutrons, resulting in a total fluence of 1.6 × 10(12) neutrons per cm(2) (±7%). Significant suppression of tumor growth was observed in mice given BNCT vs. control mice (only 424% increase in tumor volume at 14 d post irradiation vs. 1551% in untreated controls). In a separate experiment in which mice were given a second injection/irradiation treatment 7 d after the first, the tumor growth was vastly diminished (186% tumor volume increase at 14 d). A similar response was obtained for mice irradiated for 60 min (169% increase at 14 d), suggesting that neutron fluence was the limiting factor controlling BNCT efficacy in this study.

Resveratrol regulates pathologic angiogenesis by a eukaryotic elongation factor-2 kinase-regulated pathway.

Abnormal angiogenesis is central to the pathophysiology of diverse disease processes including cancers, ischemic and atherosclerotic heart disease, and visually debilitating eye disease. Resveratrol is a naturally occurring phytoalexin that has been demonstrated to ameliorate and decelerate the aging process as well as blunt end organ damage from obesity. These effects of resveratrol are largely mediated by members of the sirtuin family of proteins. We demonstrate that resveratrol can inhibit pathological angiogenesis in vivo and in vitro by a sirtuin-independent pathway. Resveratrol inhibits the proliferation and migration of vascular endothelial cells by activating eukaryotic elongation factor-2 kinase. The active kinase in turn phosphorylates and inactivates elongation factor-2, a key mediator of ribosomal transfer and protein translation. Functional inhibition of the kinase by gene deletion in vivo or RNA as well as pharmacological inhibition in vitro is able to completely reverse the effects of resveratrol on blood vessel growth. These studies have identified a novel and critical pathway that promotes aberrant vascular proliferation and one that is amenable to modulation by pharmacological means. In addition, these results have uncovered a sirtuin-independent pathway by which resveratrol regulates angiogenesis.

Interleukin-10 overexpression promotes Fas-ligand-dependent chronic macrophage-mediated demyelinating polyneuropathy.

BACKGROUND: Demyelinating polyneuropathy is a debilitating, poorly understood disease that can exist in acute (Guillain-Barré syndrome) or chronic forms. Interleukin-10 (IL-10), although traditionally considered an anti-inflammatory cytokine, has also been implicated in promoting abnormal angiogenesis in the eye and in the pathobiology of autoimmune diseases such as lupus and encephalomyelitis. PRINCIPAL FINDINGS: Overexpression of IL-10 in a transgenic mouse model leads to macrophage-mediated demyelinating polyneuropathy. IL-10 upregulates ICAM-1 within neural tissues, promoting massive macrophage influx, inflammation-induced demyelination, and subsequent loss of neural tissue resulting in muscle weakness and paralysis. The primary insult is to perineural myelin followed by secondary axonal loss. Infiltrating macrophages within the peripheral nerves demonstrate a highly pro-inflammatory signature. Macrophages are central players in the pathophysiology, as in vivo depletion of macrophages using clodronate liposomes reverses the phenotype, including progressive nerve loss and paralysis. Macrophage-mediate demyelination is dependent on Fas-ligand (FasL)-mediated Schwann cell death. SIGNIFICANCE: These findings mimic the human disease chronic idiopathic demyelinating polyneuropathy (CIDP) and may also promote further understanding of the pathobiology of related conditions such as acute idiopathic demyelinating polyneuropathy (AIDP) or Guillain-Barré syndrome.

Interleukin-10 promotes pathological angiogenesis by regulating macrophage response to hypoxia during development.

Aberrant angiogenesis in the eye is the most common cause of blindness. The current study examined the role of interleukin-10 (IL-10) in ischemia-induced pathological angiogenesis called neovascularization during postnatal development. IL-10 deficiency resulted in significantly reduced pathological retinal angiogenesis. In contrast to the choroicapillaris where IL-10 interferes with macrophage influx, IL-10 did not prevent anti-angiogenic macrophages from migrating to the retina in response to hypoxia. Instead, IL-10 promoted retinal angiogenesis by altering macrophage angiogenic function, as macrophages from wild-type mice demonstrated increased vascular endothelial growth factor (VEGF) and nitric oxide (NO) compared to IL-10 deficient macrophages. IL-10 appears to directly affect macrophage responsiveness to hypoxia, as macrophages responded to hypoxia with increased levels of IL-10 and STAT3 phosphorylation as opposed to IL-10 deficient macrophages. Also, IL-10 deficient macrophages inhibited the proliferation of vascular endothelial cells in response to hypoxia while wild-type macrophages failed to do so. These findings suggest that hypoxia guides macrophage behavior to a pro-angiogenic phenotype via IL-10 activated pathways.